TOR, the "target of rapamycin" is a giant protein kinase that is critical to the regulation of cell growth in response to nutrient and energy sufficiency, to growth factors (such as insulin and IGF-1) and to other developmental signals. Rapamycin inhibits a subset of TOR's actions that underlie cell growth, and in some cell backgrounds, inhibits proliferation. Rapamycin is in clinical use because of its immunosuppressant action and antiproliferative effects on vascular smooth muscle cells; it is also under evaluation as an anti-cancer agent. Two sets of recent discoveries have provided important insight into TOR function and regulation, and work supported by this award has contributed to both areas. Biochemical methods established that TOR functions in two physically independent multiprotein complexes, only one of which (called TOR complex 1; TORC1) is inhibitable by Rapamycin. The interaction of TORC1 with its known substrates through raptor has been elucidated. TOR complex 2 is resistant to rapamycin, and appears to regulate the actin cytoskeleton through unknown effectors. TORC2 also serves as a necessary activating kinase (so called PDK2) for Akt, whereas TORC1 acts as a feedback inhibitor of Akt. Independently, the molecular connection between the insulin/IGF-1 receptors/Type 1 PI-3Kinase/Akt and TORC1 was shown to be the Tuberous Sclerosis heterodimer complex (TSC1/2) and Rheb, a Ras-like GTPase, Rheb is a positive regulator of mTOR signaling to TOR complex 1, that acts in part directly on TORC1. TSC1/2 is an activator of Rheb GTPase, thereby inhibiting Rheb; insulin/IGF-1, through Akt,and other inputs through the MAPK pathway suppress TSC GAP function, thereby promoting TORC1 signaling. Depletion of amino acids, especially leucine, inhibits TORC1 signaling, mostly independent of TSC1/2, but in a manner that is rescued by overexpressed Rheb. We find that leucine withdrawal disrupts'the interaction between Rheb and TOR. We propose to carry out a structure-function analysis of Rheb so as to understand how its interactions within TOR1 control TORC1 signaling, and define the biochemical mechanism by which leucine sufficiency controls the Rheb- mTOR interaction in vivo. We will characterize the transcriptional responses to Rheb-GTP, TORC1 and TORC2 and identify and characterize additional candidate Rheb effectors. In addition, we will elucidate the operation of TORC2, by defining the physical and functional interactions among the components unique to TORC2, the regulatory inputs that control TORC2 signaling in vivo, and the identity of additional TORC2 targets/substrates. The results of these studies will provide basis for therapeutic interventions in this pathway, which is crucial to both human cancers and to diseases like Type 2 diabetes